TYPE 6 SECRETION

In situ architecture, function, andevolution of a contractileinjection systemDésirée Böck,1* João M. Medeiros,1* Han-Fei Tsao,2 Thomas Penz,2† Gregor L. Weiss,1

Karin Aistleitner,2‡ Matthias Horn,2§ Martin Pilhofer1§

Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophagetail–like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS)is defined by intracellular localization and attachment to the cytoplasmic membrane.Here we used cryo-focused ion beam milling, electron cryotomography, and functionalassays to study a T6SS in Amoebophilus asiaticus. The in situ architecture revealed threemodules, including a contractile sheath-tube, a baseplate, and an anchor. All modulesshowed conformational changes upon firing. Lateral baseplate interactions coordinatedT6SSs in hexagonal arrays.The system mediated interactions with host membranes andmay participate in phagosome escape. Evolutionary sequence analyses predicted thatT6SSs are more widespread than previously thought. Our insights form the basis forunderstanding T6SS key concepts and exploring T6SS diversity.

Contractile injection systems (CISs) delivereffectors tomediate bacterial cell-cell inter-actions. Their structural components arehomologous to the contractile tails of phages(1). CISs consist of an inner tube surrounded

by a contractile sheath, a spike capping the innertube, and a baseplate complex at the base of thesheath. Sheath contraction propels the inner tubeinto the target. Two modes of action divide CISsinto “extracellular CISs” (eCISs) and “type 6 se-cretion” (T6S) systems (T6SSs). eCISs resembleheadless phages; they are released into themediumand bind to the target cell surface. Examples areantibacterialR-typebacteriocins (2), insecticidal anti-feeding prophages (Afps) (3), and metamorphosis-inducing structures (MACs) (4). By contrast, theT6SS is defined by its cytoplasmic localization andanchoring to the inner membrane (5–9).Amoebophilus asiaticus (hereafter referred to

as Amoebophilus or amoebophili) is an obligateintracellular bacterial symbiont of amoebae (10).TheAmoebophilusgenomedoesnot encodeknownsecretion systems (11); however, it contains a genecluster with similarities to that of Afps (12). Wereasoned that the Afp-like gene cluster might en-code a system that would give insight into T6SSstructure, function, and evolution.To investigate whether Amoebophilus produced

any CISs, we imaged bacterial cells that were pu-rified from infected amoeba cultures by electroncryotomography (ECT). Fifty percent of the imagedcells (n = 92) contained phage tail–like assemblies.Like T6SSs and unlike eCISs, the structures were

always located in theAmoebophilus cytoplasm andattached to the cytoplasmic membrane. The struc-tures were always found in bundles of 2 to 34 par-allel individual systems (8 on average; Fig. 1, A toC;fig. S1, A toD; andmovie S1). Cells contained eitherone or two (85%) bundles (Fig. 1D and fig. S1E).Inside a bundle, the structures were arranged inordered hexagonal arrays (Fig. 1, E andF). Extendedand contracted conformational states could be dis-tinguished by differences in length (242 ± 7 nm, n =254, and 122 ± 6nm,n= 153, respectively), diameter(14 ± 2 nmand 19 ± 2 nm, respectively), and surfaceproperties of the sheath (helical ridges on thecontracted structures) (Fig. 1, B and C, and fig. S1,F to I). The narrow distribution of sheath lengthsindicates a mechanism for length control. In ad-dition, not all structures inside an array appearedto fire together (Fig. 1, B to D and F, and fig. S1E).The arrays of contractile structures were en-

coded by the Amoebophilus Afp-like gene cluster.Twelve of its components were detected in asheath preparation (table S1). Sheath structureswere labeled by specific antibodies (fig. S1, J andK). Furthermore, tomograms of purified sheathrevealed contracted sheaths whose structure (fig.S1, L and M) and dimensions (length 115 ± 7 nm,diameter 19 ± 2 nm, n = 51) were consistent withthe structures observed in situ (fig. S1, F to I).To observe macromolecular details, we aver-

aged subvolumes of extended T6S-like machines(Fig. 2, A to K, and movies S2 and S3). The struc-ture revealed three major modules: a sheath-tube assembly, a baseplate, and an anchoringcomplex (Fig. 2, A to I). This segmentation wassupported by the comparison of the average withthe structure of the minimal composition of acontractile injection system derived from the T4phage tail (13) (Fig. 2B). All threemodules showedsixfold rotationally symmetric features (fig. S2).Densities for the inner tube (7 nm diameter) andsheath (14 nmdiameter) could be clearly discerned(Fig. 2, A and I). The baseplate was overall hexag-

onal (Fig. 2, G and H) and established connectionswith baseplates of neighboring structures, therebycoordinatingmultiple systems in hexagonal arrays(Fig. 2, J and K). The central baseplate regionfeatured additional densities that reinforced ina threefold symmetrized average (fig. S2, I to M).The anchoring complex consisted of a six-footedplatform that attached the baseplate to the innermembrane (Fig. 2, A andD to F). Densities whosedimensions were consistent with a spike complex(14) were seen capping the inner tube and pro-truded through the centers of baseplate andanchor (Fig. 2, A and B and E to G). The averageslacked densities thatwould indicate the presenceof an elaborate trans-envelope complex (Fig. 2Aand fig. S2, A and E to G) such as TssJLM (9).The T4 phage baseplate triggers sheath con-

traction by a large-scale conformational change(13). We therefore calculated an average of con-tracted structures (Fig. 2, L to R, and movies S4and S5). Again, sheath, baseplate, and anchormodules were identified (Fig. 2, L to R). All threemodules revealed pronounced conformationalchanges as compared to the extended state (movieS6). Similar to the Vibrio cholerae T6SS (5), thesheath diameter increaseduponcontraction, alongwith the appearance of helical surface ridges andthe disappearance of the inner tube (Fig. 2, I andR). The baseplate showed a widening and a loss ofdensities in the center (Fig. 2, G and H and P andQ). Likewise, the anchoring platform showedlateral expansion (distance between opposingfeet at the inner membrane increased from 16 to19 nm) and a loss of the spike density in thecenter (Fig. 2, E and F and N and O). On a largerscale, the spacing between contractile structuresincreased from 22 nm between extended struc-tures to 30 nm between contracted structures.We then tested whether the Amoebophilus

system secreted tube protein into extracellularspace. Immunoblotting detected tube protein(Hcp), but no sheath, in the supernatant of aculture (fig. S3), indicating Hcp translocation.The system thus fulfills the functional hallmarkof canonical T6SSs. Together with the structuraldata, this suggests that the Amoebophilus Afp-likegene cluster encodes a T6SS rather than an eCIS.Next, we investigated the function of T6SS ar-

rays. Amoebophili were internalized in the first2 hours postinfection (hpi) and exited the amoe-bae ~144 hpi (fig. S4). To observe intracellularamoebophili by ECT, we used cryo-focused ionbeammilling to generate lamellae that were suit-able for ECT (fig. S5) (15, 16). At 0.25 hpi, coccoidamoebophili were inside phagosomes (80%, n =20; Fig. 3A and movie S7). At later stages, mostamoebophili had escaped into the cytosol (94%,n = 94), differentiated into rods, and replicated(Fig. 3, B to D; fig. S6A; and movies S8 to S10).The sheath mRNA level was 230-fold higher inextracellular amoebophili compared to the rep-licative stage (table S2). Likewise, cryotomo-grams of amoebophili from different infectionstages showed that T6SSs were most abundantin extracellular amoebophili (58%, n = 19) andat early infection stages (1 hpi, 96%, n = 25; 2 hpi,69%, n = 13), whereas replicative amoebophili

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1Institute of Molecular Biology and Biophysics,Eidgenössische Technische Hochschule Zürich, CH-8093Zürich, Switzerland. 2Division of Microbial Ecology, Universityof Vienna, A-1090 Vienna, Austria.*These authors contributed equally to this work. †Present address:CeMM Research Center for Molecular Medicine of the AustrianAcademy of Sciences, 1090 Vienna, Austria. ‡Present address:National Institute of Allergy and Infectious Diseases, Hamilton, MT59840, USA. §Corresponding author. Email: horn@microbial-ecology.net (M.H.); pilhofer@biol.ethz.ch (M.P.)

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Böck et al., Science 357, 713–717 (2017) 18 August 2017 2 of 5

Fig. 1. The Amoebophilus Afp-like gene cluster encodes orderedarrays of T6S-like structures. (A to C) Cryotomograms of purifiedAmoebophilus cells revealed bundles of cytoplasmic, membrane-bound,contractile structures in extended (“E”) and contracted (“C”) conforma-tions. CP, cytoplasm; IM, inner membrane; OM, outer membrane. Shownare three different 14-nm slices through the same tomogram. (D) Bundles

comprised 2 to 34 individual machines (green, extended; yellow,contracted) and were organized in one or two bundles per cell. Shown isa model of the tomogram shown in (A) to (C). Blue, outer membrane;cyan, inner membrane. (E and F) Structures were arranged in hexagonalarrays (lattice indicated in orange). Shown are 15-nm (E) and 8-nm (F)cross-sectional slices. Bars: 100 nm.

Fig. 2. The T6SS in situ architecture reveals three major modules,conformational changes upon firing, and the structural basis of arrayformation. Subtomogram averages of extended (A to K) and contracted(L to R) T6SSs revealed three major cytoplasmic modules: Sheath-Tube,Baseplate, and Anchor (indicated by brackets). Shown are 0.81-nm [(A)and (B), (E) to (I)] and 1.38-nm [(L), (N) to (R)] longitudinal [(A), (B), and (L)]and perpendicular [(E) to (I), (N) to (R)] slices through sixfold rotationallysymmetrized averages, and their three-dimensional models [(C), (D), (J),(K), and (M)].The positions of perpendicular sections are indicated in (A) and(L). OM, outer membrane; IM, inner membrane. Bars: 10 nm [(A) to (D), (L),and (M) to scale; (E) to (I), (N) to (R) to scale]. The segmentation in threemodules was supported by the overlay (B) with the structure of the minimalcomposition of a contractile injection system derived from the T4 phage tail

[from (13)]. It allowed the putative assignment of densities correspondingto tube (gp19/gp48/gp54), sheath (gp18), spike complex (gp5/gp5.4/gp27),and baseplate wedge components (gp6/gp7/gp25/gp53). Densities that werenot accounted for were assigned to the anchor module [segmented in(D); white, anchor; orange, spike]. Upon firing, pronounced conformationalchanges were detected in all modules [movie S6 shows a morph betweenmodels shown in (C) and (M)]. The sheath increased in diameter andformed surface ridges [(A), (C), (I), (L), (M), and (R)]. Baseplate and anchorshowed widening and loss of densities in the center [(A), (C), (E) to (H),(L), (M), (N) to (Q)]. Ordered arrays were established by lateral interactionsof the hexagonal baseplates [(J) and (K)]. Shown are top (J) and side(K) views of a model that was assembled from masked averages. For viewingpurposes, two different baseplate levels are colored in purple and orange.

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did usually not harbor structures (5%, n = 20)(Fig. 3E and fig. S6, B to G). The process of exitingthe phagosome during early infection (up to 2 hpi)correlated with increased fractions of contractedstructures (Fig. 3E). Experiments comparing thepotential of amoebophili from different infectionstages to establish new infections showed thathost infection rates positively correlated with T6SSexpression (fig. S6H). Likewise, we tested amoe-bophili from different infection stages for hemo-lytic activity and found that red blood cell (RBC)lysis alsopositively correlatedwithT6SS expression(fig. S7). ECT imaging of amoebophili that had thepossibility to interact with RBCs showed a 30%increased fraction of contracted structures, com-pared to a control sample (Fig. 3F;P<0.0001). Theanalysis of tomograms of purified amoebophilithat were found inside phagosomes (39% at 1 hpi)

revealed that any contact site between the phago-somemembrane and the outermembrane of thebacterium correlated with the presence of T6SSs(with at least one contracted structure) (n = 14;Fig. 3, G and H; fig. S8; andmovie S11). Together,our data suggest that T6S arrays mediate inter-actionswith hostmembranes andmay participatein phagosome escape. It remains open whetherphagosome rupturing ismediated bymechanicalforces or membrane-targeting effectors.Next, we sought to understand the evolution-

ary history of the Amoebophilus Afp-like genecluster. We compared three key components(sheath, tail tube, baseplate component gp25) toother CISs (table S3). Similarities were highestwith a gene cluster of unknown function in therelated symbiont Cardinium hertigii (17). Mod-erate similarities were found for Afps andMACs,

both mediating interactions with animal larvae(3, 4). Lowest (or no similarities at all) were de-tected for T6SSs [subtypes i, ii, iii (18)] and con-tractile phages. Likewise, phylogenetic analysesrevealed that Amoebophilus (and Cardinium) se-quences stably clustered in a monophyletic groupwith Afps andMACs, rather thanwith T6SSi-iii (Fig.4A and fig. S9). With the exception of gp7, theanalysis of gene content detected Amoebophilushomologs of all components that are conservedacross CISs and phages (13). These include puta-tive sheath (gp18/TssBC), inner tube (gp19/Hcp,gp48, gp54), spike (gp5/VgrG, gp5.4/PAAR, gp27),and three baseplate wedge components (gp6/TssF, gp25/TssE, gp53) (Fig. 4B and table S4).The lack of gp7might be explained by the presenceof two gp6 homologs, and the suggestion that gp6and gp7 diverged from a common ancestor (13, 19).

Böck et al., Science 357, 713–717 (2017) 18 August 2017 3 of 5

Fig. 3. T6S arrays are required during early infection stages andmediate interactions with host membranes. (A to D) Bacteria inside theirhost were imaged by cryo-focused ion beam milling and ECT (fig. S5). At0.25 hpi, most coccoid amoebophili (white arrowheads) were found insidephagosomes (P). At later time points (0.5 to 2 hpi), amoebophili hadescaped into the amoeba cytosol (aC). Amoebophili differentiated into rods(white arrows) and divided. A small fraction did not escape, showing signsof degradation (black arrowhead). Shown are 15-nm slices throughcryotomograms. Asterisk, T6S array; g, golgi apparatus; m, mitochondrion.Bars: 100 nm. (E) Abundance of T6S arrays was determined by ECT ofamoebophili purified from synchronized cultures, and found to be highestin extracellular “EC (144 hpi)” and early intracellular infection stages (1 hpi,2 hpi). The increase in the contraction rate between 1 and 2 hpi correlatedwith the escape from the phagosome. Shown are the percentages of cellswith T6S arrays (red), percentages of T6S structures that were contracted

(black), and percentages of cells found inside phagosomes (blue). T6Sarrays, number of quantified amoebophili: n1hpi = 25, n2hpi = 13, n72hpi = 20,n144hpi = 15, nEC(144 hpi) = 19; Contraction, number of quantified T6Sstructures: n1hpi = 168, n2hpi = 88, n72hpi = 4, n144hpi = 4, nEC(144 hpi) = 59;Inside Phagosome, number of quantified amoebophili: n1hpi = 121, n2hpi = 118,n72hpi = 218, n144hpi = 337, nEC(144 hpi) = 55). (F) Amoebophili showedhemolytic activity (fig. S7). Extracellular amoebophili that interacted withRBCs showed an increased T6S contraction rate (****P < 0.0001; nRBC

–

=506; nRBC

+

= 480; mean ± SEM). (G and H) Amoebophili residing inphagosomes revealed contact sites (black arrowhead) between theAmoebophilus outer membrane and the phagosome. Any such contact sitecorrelated with a T6S array (n = 14). Shown are a 15-nm tomographic slice(G) and the corresponding model (H). P/red, phagosome; OM/blue, outermembrane; IM/cyan, inner membrane; CP, cytoplasm; E/green, extendedT6SS; yellow, contracted T6SS; Bars: 100 nm.

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Components that are exclusively conserved in ca-nonical T6SSs (and absent in eCISs/phages) werenot found, including TssJLM [trans-envelope com-plex (9)] or ClpV [sheath recycling (20)]. By con-trast, the Amoebophilus cluster encodes proteinsthat were thought to be specific for eCISs and con-tractile phages. The length of the AmoebophilusT6SSs, for instance, is likely controlledbyAasi_1072/1806, which are homologs of tail terminator andtape measure proteins in Afps and phages (21, 22).Indeed, sheath length can be predicted from TmPsequence (22) and correlates well with the lengthof Amoebophilus sheaths (fig. S10). Another ex-ample is an Rz-like endopeptidase (Aasi_1068),which usually co-occurs with a holin to mediatethe release of eCISs and phages from the bacte-rial cytoplasm (3, 23).In conclusion, our structural and functional

data showed that the Amoebophilus Afp-like genecluster encodes a T6SS, whereas sequence anal-yses indicated a close relationship to eCISs. Wetherefore introduce the term “T6SS subtype 4”(T6SSiv). In contrast to the distant relationshipsof T6SSi-iii to eCISs and phages that obstruct thereconstruction of an evolutionary path (1, 24), wecan hypothesize that T6SSiv evolved from an Afp/

MAC-like eCIS (independently of T6SSi-iii) by theloss of tail fibers, loss of holin, and the estab-lishment of interactions with the cytoplasmicmembrane. Alternatively, T6SSiv represents aprimordial system from which eCISs, phages,and T6SSi-iii have evolved (Fig. 4C). Both scenar-ios predict that T6SSs are more abundant thanpreviously thought. Indeed, T6SSiv-like gene clus-ters were detected in six diverse bacterial phyla(table S5). The finding that diverse T6SS sub-types do not share a conserved gene set thatwould distinguish them from eCISs or phagesemphasizes the necessity of an integrative ap-proach to discover and characterize new systems.

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ACKNOWLEDGMENTS

We thank F. Bosia, A. Harreither, P. Gasser, S. Rutz, P. Szwedziak,P. Tittmann, R. Wepf, and C. Zaubitzer for technical support. ScopeMis acknowledged for instrument access at ETHZürich.We thankO.Medaliafor microscope access at the University of Zürich. T. Ishikawa, R. Wepf,

Böck et al., Science 357, 713–717 (2017) 18 August 2017 4 of 5

Fig. 4. Amoebophilus T6SSiv is closely related to eCIS. (A) Phylogeneticanalyses of sheath protein sequences showed that T6SSi-iii formed amonophyletic group with high support (bootstrap supports are indicated atnodes). Amoebophilus T6S sheath, however, stably clustered in a group withthe sheath from a gene cluster inCardinium (structure and function unknown),and with the eCIS sheaths of Afp and MAC.The marked node was stable in allcalculated trees as determined with different treeing methods and differentcomponents (fig. S9). (B) The Amoebophilus Afp-like gene cluster encodes allcomponents that are conserved (blue) among all contractile injection systems

(canonical T6SS/eCIS/phages), although it lacks any homologs of compo-nents that are specific for canonical T6SSs (yellow). Instead, the clusterharbors genes that are typical of eCISs and phages (red). Shown are twoAmoebophilus genomic regions, locus tags, and gene annotations. See alsotable S4. (C) Schematic showing the major components of canonical T6SS,Amoebophilus T6SSiv, and eCIS (homologs in same colors).T6SSiv evolvedeither from eCISs, or alternatively,T6SSiv represents a primordial system thatgave rise to eCISs/phages/T6SSi-iii. Both scenarios predict that T6SSs aremore abundant than previously estimated (table S5).

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and Pilhofer Lab members are acknowledged for discussions.R. Kostanjsek is acknowledged for generating preliminary data.W.-D. Hardt, H. Hilbi, R. Kooger, V. Panse, N. Shikuma, M. Swulius,and E. Tocheva are acknowledged for comments on the manuscript.We thank N. Taylor for providing us with the T4 model shown in Fig. 2B.Preliminary observations were made in the lab of G. J. Jensen. M.P.is supported by the European Research Council, the Swiss NationalScience Foundation, and the Helmut Horten Foundation. M.H. is

supported by the European Research Council (ERC StG, no. 281633)and Austrian Science Fund (I 1628-B22). Subtomogram averages andtomograms were deposited in the Electron Microscopy Data Bank(accession numbers EMD-3791 and EMD-3793 to EMD-3801).

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Materials and MethodsFigs. S1 to S11Tables S1 to S5References (25–43)Movies S1 to S11

19 May 2017; accepted 17 July 201710.1126/science.aan7904

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In situ architecture, function, and evolution of a contractile injection system

PilhoferDésirée Böck, João M. Medeiros, Han-Fei Tsao, Thomas Penz, Gregor L. Weiss, Karin Aistleitner, Matthias Horn and Martin

DOI: 10.1126/science.aan7904 (6352), 713-717.357Science 

, this issue p. 713Scienceorganized in multibarrel gun-like arrays and may contribute to the survival of bacteria inside their host.cellular context. They identified three modules and showed large-scale structural changes upon firing. T6SSs aremodern electron microscopy methods and functional assays to resolve the structure and function of a T6SS in the

usedet al.bacteriophages. The so-called type 6 secretion system (T6SS) functions from inside a bacterial cell. Böck To interact with other cells, bacteria use contractile machines that function similarly to membrane-puncturing

Identification of a new injection system

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